Method of cancer screening primarily utilizing non-invasive cell collection, fluorescence detection techniques, and radio tracing detection techniques

ABSTRACT

A cancer screening method is provided, wherein the method is characterized by administering a compound to a patient, the compound being a complex of a fluorescent marker and a radioactive marker, in a dose emitting between 5 Gy and 20 Gy radiation. Cells are then collected preferably through non-invasive or minimally invasive means. If fluorescence is observed in the exfoliated cells, tomographic scanning is conducted to further locate and/or confirm suspected malignant areas or metastatic areas. Further observation or treatment may be conducted either through fluorescence guided endoscopy, photo-dynamic therapy, and/or radiation treatment.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 10/176,558 filed Jun. 21, 2002 which is acontinuation-in-part of co-pending PCT Application Serial No.PCT/US00/34121 filed on Dec. 16, 2000, which is a continuation of U.S.patent application Ser. No. 09/472,945, filed on Dec. 27, 1999, now U.S.Pat. No. 6,190,877.

TECHNICAL FIELD

[0002] This invention relates to a method of cancer screening and, moreparticularly, to a method primarily utilizing non-invasive cellcollection techniques, and a combination of fluorescence detectiontechniques and radio tracing techniques for positive identification ofmalignant cells.

BACKGROUND ART

[0003] There are a number of prior art methods and apparatuses which areused in the detection and treatment of cancer. Fluorescent markers havebeen used to help identify cancerous tissue within a patient. Radiotracers or markers have also been used in the detection and treatment ofcancer. There are also a number of prior art methods and apparatuseswhich relate to flow cytometry and the act of segregating and countingmalignant cells within a tissue sample.

[0004] One example of a prior art reference which discloses the use offluorescence detection for cancer screening is U.S. Pat. No. 5,270,171to Cercek, et al. This reference teaches a method to identify, separateand purify the factor or factors that provoke a response by SCM(structuredness of the cytoplasmic matrix) responding lymphocytes. Theuse of such purified factor or factors enhances a SCM cancer screeningtest. The SCM is a peptide of at least nine amino acid residues. Theresidues produce at least a 10% decrease in the intracellularfluorescence polarization value of SCM responding lymphocytes fromdonors afflicted with cancer. Antibodies specific for the SCM factor areuseful in immunoassays that can detect the factor, including detectionof cancer cells grown in vitro. The SCM factor is useful for screeningof blood samples and other body fluids or cell aspirates for thepresence of malignancy in the donor. A method is also disclosed fortesting lymphocytes obtained from the donor for presence or absence of amalignancy. A further method is also disclosed for screening a bloodsample for the presence of a malignancy in a body of a donor.

[0005] U.S. Pat. No. 5,391,547 discloses a method of using porphyrins todetect lung cancer, and more particularly, to the use of tetra-arylporphyrins. The porphyrins are used as a fluorescent tracer for cancersof the lung. The porphyrins may be complexed with Copper 64 (⁶⁴Cu) orCopper 67 (⁶⁷Cu). Thus, the complex can be used as radio tracers aswell. The ⁶⁷Cu provides a source of beta radiation for selectivedestruction of lung malignancies as well as gamma radiation useful forimage analysis, as by single photon emission compute tomography. The⁶⁴Cu may be used for radio tracing wherein a positron emissiontomography technique can be used to locate the malignant tissue.

[0006] U.S. Pat. No. 5,562,114 to Chu, et al. discloses a diagnosticimmunoassay method using monoclonal antibodies. These monoclonalantibodies are capable of identifying an antigen associated withcarcinomas of ductal lineage and can be used both diagnostically andtherapeutically. More specifically, the monoclonal antibodies of thisreference are capable of targeting the breast carcinoma cells in vivo.The monoclonal antibodies are purified and are labeled with radioactivecompounds, for example, radioactive iodine, and then are administered toa patient intravenously. After a localization of the antibodies at thetumor site, they can be detected by emission, tomographical and radionuclear scanning techniques thereby pinpointing the location of thecancer.

[0007] U.S. Pat. No. 5,087,636 to Jamieson, et al. discloses a method toidentify and destroy malignant cells in mononuclear cell populations.This method includes the steps of contacting a composition of bonemarrow cells or other cells with a green porphyrin of a specificcompound, irradiating the cell composition with light at a wave lengtheffective to excite fluorescence of the green porphyrin, and thendetecting the presence or absence of fluorescence indicating malignancy.This reference also discloses the steps by which the bone marrow cellsare removed, separated, washed and diluted to an appropriateconcentration for treatment, incubated, centrifuged, and exposed to theirradiating light.

[0008] U.S. Pat. Nos. 5,308,608 and 5,149,708 to Dolphin, et al.disclose specific types of porphyrin compounds which may be used fordetection, photosensitization, or the destruction of a targetedbiological material when the targeted tissue is contacted with thespecified porphyrin, and irradiated with light that excites thecompound.

[0009] U.S. Pat. No. 5,211,938 to Kennedy, et al. discloses a method ofdetection of malignant and non-malignant lesions by photo chemotherapyof protoporphyrin IX precursors. 5-amino levulinic acid (5-ALA) isadministered to the patient in an amount sufficient to induce synthesisof protoporphyrin IX in the lesions, followed by exposure of S thetreated lesion to a photo activating light in the range of 350-640nanometers. Naturally occurring protoporphyrin IX is activatable bylight which is in the incident red light range (600-700 nanometers)which more easily passes through human tissue as compared to light ofother wave lengths which must be used with other types of porphyrins. Inshort, the use of 5-ALA makes cell fluorescence easier to observe, andalso greatly reduces the danger of accidental phototoxic skin reactionsin the days following treatment since protoporphyrin IX precursors havea much shorter half life in normal tissues than other popularly usedporphyrins.

[0010] Another set of references exists which relate to flow cytometryutilizing fluorescence producing compounds. One such prior art referenceincludes U.S. Pat. No. 5,605,805 to Verwer, et al., which discloses amethod for determining the lineage of acute leukemia cells in the sampleby fluorocytometry. Other examples of fluorocytometry utilizingfluorescence include U.S. Pat. No. 5,418,169 to Crissman, et al., U.S.Pat. No. 5,556,764 to Sizto, et al., and U.S. Pat. No. 5,627,040 toBierre.

[0011] Present methods relating to cancer screening using fluorescencedetection systems require the use of interventional devices such asendoscopes which have the special capability of delivering specifiedlight frequencies to a targeted area within a patient. These endoscopesilluminate the targeted part of the body in which cancer is suspected.The light delivered at a specified frequency illuminates an area whichhas previously been subjected to some type of fluorescent marker, suchas a porphyrin which causes malignant cells to illuminate or fluoresceunder observation of light at a specified frequency. In all cases,introduction of an endoscope into the body requires some type ofsedation or general or local anesthesia. Once a tumor has been locatedby use of the interventional device, depending upon the type of tumor,photo chemotherapy or other treatment means can be used. However, priorto actual treatment, there must be a confirmed test of cancer.Accordingly, the tumor still needs to be sampled by an appropriatebiopsy method. Generally, biopsy methods also require some type ofsedation or anesthesia. Thus, traditional methods of confirming amalignancy may require at least two interventional surgical procedures.

[0012] While each of the foregoing references may be adequate for theirintended purposes, many of these inventions require surgical techniquesto remove the cell samples which can be traumatic to the patient.Furthermore, many of the references require complex equipment, andspecial medical expertise in order to conduct the procedures and to makethe diagnoses. Therefore, there is a need for a reliable cancerscreening technique or method which can test for cancer in a widevariety of cells and which may be accomplished by non-invasive orminimally invasive cell collection techniques which limit patienttrauma, are inexpensive to conduct, and can be confirmed positively by apathologist, oncologist or other physician without additional testing orscreening. There is also a need for a reliable cancer screeningtechnique or method which can utilize two different types of screeningtechnologies within a single cancer screening procedure, thereforeimproving the ability to make a diagnosis of cancer, and also to providea follow-on treatment of the cancer, either through photo-dynamictherapy and/or through radiation-treatment. The invention describedbelow provides each of these advantages, among others, which will beapparent to those skilled in the art.

DISCLOSURE OF THE INVENTION

[0013] The present invention relates to a method of cancer screeningutilizing non-invasive or minimally invasive cell collection techniques,fluorescence detection techniques, and radio detection techniques. Theinvention herein also contemplates treatment of a cancer by variousphoto therapy treatments and/or radiation treatments.

[0014] The term “non-invasive” as used herein and as applied to aspecific cell collection technique shall mean cell collection which doesnot involve the forced removal of tissue as by the act of cutting orotherwise tearing away cell tissue which would normally remain attachedto the body. As discussed further below, a cell collection techniqueusing a cytological brush would be considered non-invasive because,although contact is made with a targeted area of tissue to be removed,the cytological brush simply removes a top layer(s) of cells which wouldnormally exfoliate or desquamate from the body. Thus, a cytologicalbrush used according to the cell collection techniques of this inventiondoes not involve the scraping of tissue to a degree that it cuts ortears tissue away from the body. The term “minimally invasive” as usedherein and as applied to other cell collection techniques disclosedherein shall mean the removal of cells from the body which requires someinterventional means for accessing the targeted group of cells, but doesnot require the actual tearing or cutting away of such targeted tissue.As discussed further below, minimally invasive cell collectiontechniques include the use of a fine gauge needle or catheter which mustpenetrate the body to gain access to interior targeted tissue. Thisminimally invasive cell collection technique is used specifically withthe collection of cells from the central nervous system, peritonealcavity, and thoracic cavity. Cell collection from these areas in thebody is not achieved by cutting or tearing away the tissue, but isachieved by non-invasive means once the minimally invasive accessprocedure has taken place.

[0015] Thus, according to one aspect of the present invention, theexfoliation or dislodgement of cells from the human body is achievedthrough non-invasive means. For dislodgement of pulmonary system cells,techniques are disclosed which include fist percussion while a patientis placed in a postural drainage position. For exfoliation ofgastrointestinal cells, the techniques include lavage cytology by oraladministration of a first balanced electrolyte solution to cleanse thebowel which is followed by oral administration of an additionalelectrolyte solution to produce a clear anal effluent for cytologicevaluation. Cells in the oral cavity may be collected by a cytologicalbrush. For prostate gland cell dislodgement, a physician may “milk” theprostate to express contained fluids which are carried through theductal system to the urethra via the seminal vesicles and theejaculatory ducts. For urinary tract cells, exfoliation may be achievedby rapid oral fluid intake and the use of a diuretic such as Lasix™. Forcollection of cervical and uterine cell samples, a cytological brush mayalso be used. For breast cell collection, the ductal system of thebreast may be opened by the use of a product such as Seruminex™ whichdissolves “plugs” in the ducts of the nipple, and gravity is allowed tocause fluids to drain out. The discussion below more fully details thesespecial non-invasive cell collection techniques. Collection of othercell types is also discussed below.

[0016] Once the targeted cells have been removed from the body, they areimmediately placed in a temperature controlled (37° C.) cell culturesolution or media to keep them alive a desired period of time. For mostcells, a cell transport media is used which is identical to commerciallyavailable cell culture media. A water bath is typically used to maintainthe culture at the desired temperature.

[0017] A photosensitive compound is then introduced to the cell culture.These compounds when administered in appropriate amounts selectivelyenter into pre-malignant and malignant cells, and provide a “fluorescentmarker” in the cells, primarily in the mitochondria surrounding thenucleus. The compounds which may be used in this method to inducefluorescence include 5-ALA, protoporphyrin IX, tetrakis carboxy-phenylporphine (TCPP), hematoporphyrine derivative, photofrin, and photofrinII and other known in the art to cause fluorescence in pre-malignant ormalignant cells. For TCPP, this compound enters live cells via a specialtransport mechanism found in the outer cellular wall. TCPP will notenter dead cells, thus making it important that a live cell culture bemaintained. Once inside the cell, TCPP appears to migrate to theperinuclear areas and become involved with the mitochondria. In short,the above compounds will cause pre-malignant or malignant cells tofluoresce when exposed to frequencies of light which match theexcitation frequency of the particular compound used; however healthycells will generally not fluoresce.

[0018] Once the photosensitive compounds are introduced to the cellculture, they are allowed to interact with the cell tissue a specifiedamount of time in a controlled environment. After this incubationperiod, cells may be examined by use of a fluorescence microscope to seeif any cells fluoresce. Fluorescence in the cell indicates a high degreeof suspicion for malignancy. The cell culture can first be centrifugedto help separate the cells from the cell culture fluids. The cells areresuspended in saline, and a small aliquot is placed on a slide. If nocells are found to fluoresce after initial observation under thefluorescence microscope, the cells are disaggregated and processedthrough a flow cytometer utilizing fluorescence detection. This is doneto ensure that no fluorescent cells are overlooked. Manual examinationof cell suspensions is not particularly accurate, since millions ofcells need to be examined. Flow cytometers can find a single fluorescentcell in a field of millions of cells with virtual 100% accuracy. Thefluorescence microscope and the flow cytometer provide light to matchthe excitation frequency of the particular compound used. For example,the excitation frequency for TCPP is approximately 380-450 nanometers.If fluorescent malignant cells are found by the fluorescence microscope,they may also be counted and disaggregated for further study. Afterincubation, no further care of the cell specimen is required.

[0019] Alternatively, the above-described photosensitive compounds maybe administered directly to the patient prior to cell collection. 5-ALAcan be administered orally, topically, or parenterally (by injection);however, the other compounds have to be administered topically or byinjection (parenterally). The waiting period prior to cell collection isthen two to four hours, depending upon the compound introduced. Aftersufficient time has been provided for interaction between the compoundand the targeted cells, the cells may then be exfoliated or dislodgedfrom the patient through non-invasive or minimally invasive techniques.

[0020] In addition to providing the above described photo sensitivecompounds alone, the photo sensitive compounds may be complexed with aradioisotope, such as ⁶⁴Cu or ⁶⁷Cu. Accordingly, these complexes thenprovide the ability for not only conducting fluorescence detection andphoto-therapy, but also provides the ability for observation ofmalignancies through positron emission tomography (for ⁶⁴Cu) or singlephoton emission computed tomography (for ⁶⁷Cu). Additionally, for ⁶⁷Cu,it provides a source of beta radiation for selective destruction ofmalignancies. As well understood by those skilled in the art, positronemission tomography (PET) is a scanning technique which enables precisemapping of tissue which uptakes the radioactive marker. Single photonemission computed tomography (SPECT) is closely related to PET scanningand also provides an exact means to locate targeted tissue which uptakesan introduced radio tracer.

[0021] For those cells which are not dislodged through exfoliation andare therefore left within the body, those cells are doubly tagged notonly for fluorescence, but for radioactivity as well. It may be,therefore, desirable under some circumstances to not only remove cellsfrom the body for exuluo analysis, but also to investigate believedmalignancies through fluorescence guided endoscopy, or to view thesuspect cells through the appropriate tomography technique. Oneadvantage of attaching a radioisotope to the photosensitive compounds isthat through the use of recently available tomography devices one can,in effect, conduct a complete body scan of all potentially malignantareas within the body without having to conduct separate endoscopicprocedures for each potentially malignant site found. The images takenfrom the scanning technique can then be provided to the particularspecialist for follow-on treatment, which may include surgicalintervention or radiation treatment.

[0022] By the method of this invention, a quick and reliable means isprovided for cancer screening. Because non-invasive or minimallyinvasive techniques are used for cell collection, patient trauma isreduced along with the cost of the procedure. Because the method of thisinvention provides the option of introducing the compounds ex-vivo, theconcern for any possible allergic reaction or phototoxic reaction by apatient's exposure to the sun is eliminated. Furthermore, because notissue biopsies are taken, the method of this invention eliminates theinherent hazard in administering local and/or general anesthesia. Cellmarking by use of the above-identified compounds is extremely reliablein terms of differentiating healthy cells from pre-malignant ormalignant cells. The segregation, counting and analysis of thefluorescent cells may be achieved with commercially available flowcytometers and supporting equipment. The results of the cancer screeningprocedure may be forwarded to a pathologist who may wish to conductadditional tests to further determine the exact nature of themalignancy. The fluorescing cells may be photographed to providedocumentation of malignancy.

[0023] The method of the invention also provides the option of utilizingradioactively tagged photo-sensitive compounds, wherein fluorescenceguided endoscopy can be conducted for viewing and treatment of amalignancy, and additionally, the radioactive tag allows varioustomography techniques to be used for further visualization ofpotentially malignant tissue as well as providing a means forradioactive treatment. These and other advantages are discussed morefully below in the detailed description taken in conjunction with thecorresponding figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 is a simplified flow diagram illustrating the major stepsin one aspect or part of the method of this invention;

[0025]FIGS. 2A and 2B are organizational diagrams illustrating both thetypes of cells which may be exfoliated or collected according to thecell collection techniques of this invention, and the major steps in thevarious cell collection techniques; and

[0026]FIG. 3 is another simplified flow diagram illustrating the majorsteps in another aspect of the invention including use of porphyrincomplexes for radio tracing and radiation therapy.

DETAILED DESCRIPTION OF THE INVENTION

[0027]FIG. 1 illustrates the major steps in one aspect of the method ofthis invention. As shown, the process begins with non-invasive means orminimally invasive means of cell exfoliation/collection 10. Oncecollected, the cells are then placed in a culture media or celltransport media, shown at step 12. The goal is to keep the collectedcells alive and viable after removal from the body. Thus, even at thisintermediate step, the cells should be maintained at approximately 37°C. by use of a water bath or other means. Next, the collected cells areexposed to a chemical which induces fluorescence in pre-malignant andmalignant cells, illustrated as step 14. As discussed above, thechemical compounds which are contemplated for use in this inventioninclude, but are not limited to, 5-ALA, protoporphyrin IX, TCPP,hematoporphyrin derivative, photofrin, and photofrin II. Other possiblecompounds which may be used include uroporphyrin; coproporphyren;tetraphenylporphinesulfonate (TPPS); and tetraporphen (4,N-methyulpyridil) (TMPP). These compounds, when administered inappropriate amounts, selectively enter pre-malignant and malignantcells, and provide a fluorescent marker inside the cell, primarily inthe mitochondria surrounding the nucleus. Of special utility is thecompound TCPP. This compound is available from Porphyrin Products, P.O.Box 31, Logan, Utah. As best understood, TCPP enters a live cell via aspecial cell transport mechanism in the outer cellular wall. TCPP willnot enter a dead cell. Once inside the cell, the TCPP migrates to theperinuclear areas and becomes involved with the mitochondria. TCPP isnot a stain as used in a Pap smear or other similar procedures.Therefore, it is imperative that this compound be utilized forfluorescent tagging of cells prior to cell death. For ex-vivointroduction of the chemical compound, the preferred compound is TCPP.

[0028] In terms of cell storage in the culture media, commerciallyavailable cell culture media may be used to keep the cells viable. Thesesolutions contain nutrient materials as well as selected antibiotics tocounteract any infective organisms which might alter capabilities of thecell to effectively interact with the introduced chemical. Immediatelyafter removal from the body, the cells are placed in the nutrientsolution or cell transport media. For example, the cell samples may beplaced in a commercial medium known as “Dulbecco's Modified EaglesMedium,” which is supplemented with a 10% calf serum, penicillin andstreptomycin at standard tissue culture concentrations. This medium iswell known in cell culture laboratories and readily availablecommercially or may be formulated in the cell culture laboratory bytechnicians.

[0029] After introduction of the chemical to the cell culture, theculture is incubated for a specified amount of time which allows thelive cells to absorb and interact with the compound, shown as step 16.In test trials, it has been found that a 1-4 hour incubation time isneeded for pre-malignant and malignant cells to interact with thecompounds. The average time for incubation for TCPP is 1-2 hours. Thecells are maintained in a culture incubator by use of the water bath atapproximately 37° C. The incubator may utilize air containing 5% carbondioxide which surrounds the water bath. These cell suspensions containboth non-malignant, and (in some cases) malignant cells. Normal cellsare difficult to keep alive in cell culture, and generally die in sevento 10 days from time of removal from the body regardless of the caretaken in trying to maintain their survival. Malignant cells, on theother hand, can generally be kept alive in cell culture situations forseveral weeks or months. During this early incubation period, it is alsoadvisable to agitate the cell cultures at frequent intervals to makesure that the compound comes into contact with all cells containedwithin the cell culture container.

[0030] After adequate interaction has occurred between the introducedchemical compound and the cells within the culture, the cells need to beprepared for first observation. Accordingly, the cell suspension isplaced in a centrifuge tube and the cell sample is centrifuged. Thecells become concentrated at the bottom of the centrifuge tube in a“button” of cells. The cell culture media fluid is then removed bypouring off all but about 10% of the fluid. The remaining cell culturemedia is then removed with a pipette, and the cells are resuspended in asaline solution. Small amounts of resuspended cells are then removedwith a pipette (e.g., one to two gtts) and placed on a glass slide witha slide cover. These preparation efforts are generally indicated atblock 18.

[0031] The cells are then observed under a fluorescence microscope. Thefluorescence microscope can be tuned to provide light which matches theexcitation frequency. For TCPP, the excitation frequency ranges from380-400 nanometers. The technician observing the cells under thefluorescence microscope looks for cells which fluoresce in the visiblered range (approximately 630 nanometers). The step of examining thecells and providing a matching excitation frequency for observation ofcharacteristic fluorescence is shown at block 20. If a compound otherthan TCPP is used, the fluorescence microscope is tuned to provide thematching excitation frequency, and observation is made for thecharacteristic fluorescence. There are a number of commerciallyavailable fluorescence microscopes which may be used in the method ofthis invention. Some of the manufacturers of such devices includeOlympus, Nikon, and Karl Zeiss. If any cells fluoresce, then this wouldbe an indication of malignancy. This is shown at decision point 22wherein the next step would be color photography of the fluorescingcells for documentation purposes, shown at block 23. Optionally,long-term cell observation could be conducted wherein the cells would beplaced back into a culture media for additional studies. This is shownat block 24.

[0032] If no fluorescing cells were found with the initial observationunder the fluorescence microscope at block 20, then, as also shown atdecision block 22, the cells then undergo further analysis to determinewhether there are any malignant cells present. Although the use of afluorescence microscope would allow an observer to find larger numbersof malignant cells, this is not an absolute test for finding fluorescingcells. Accordingly, these cells would be processed through a flowcytometer to find and count fluorescing cells. This is shown at block25. If the flow study reveals any fluorescing cells, this would be anindication of malignancy. As shown at block 27, the specimen could thenprepared for fluorescence activated cell sorting. Fluorescence activatedcell sorting (FACS) is done to further concentrate the specimen offluorescing cells for easier observation and is an additional functionwhich may be incorporated within a flow cytometer. Fluorescenceactivated cell sorting is achieved based upon the presence or absence offluorescence by a particular cell. By use of one of the compoundsdiscussed above, the cells which are pre-malignant or malignant willfluoresce while non-malignant cells will not. Accordingly, fluorescenceactivated cell sorting can effectively separate out in a very precisemanner pre-malignant and malignant cells versus non-malignant cells.Once the FACS study is complete, the fluorescing cells are segregatedfrom the remaining cells which do not exhibit fluorescence. Thisconcentrated sample of fluorescing cells then may be viewed under afluorescence microscope and photographed. The presence of even onefluorescing cell can be cause for making a screening diagnosis ofcancer. If no fluorescing cells are found, then the screening diagnosisis no malignancy, shown at block 28. Optionally, a long-term study ofthe fluorescing cells found at block 25 may also be conducted.

[0033] Although the above procedure describes the step of conducting aFACS study, it should be understood that, in those instances in which nofluorescing cells are found under initial observation at block 20, ascreening diagnosis of a malignancy can actually be made once the cellsare processed through the flow cytometer and fluorescent cells are foundand counted. As discussed above, the main reason that the FACS study isconducted is to better concentrate the fluorescing cells within asmaller sample size. Otherwise, finding the cells under the fluorescencemicroscope and photographing the cells would be much more difficult.

[0034] It should also be understood that the collection of cells by theminimally/non-invasive methods discussed below may result in anextremely large number of cells being collected. For example, a specimensubmitted from the uterine cervix collected by use of the cytology brushexplained below, on the average, collects approximately 5 million cells.A careful manual examination of 5 million cells would take several days.Accordingly, in instances such as these where there are such a largenumber of cells, the first observation step at block 20 would beconducted by using several slides and observing them in the fluorescencemicroscope. If no fluorescing cells were found, the remainder of thespecimen can then be processed through the flow cytometer. As desired, aFACS could also be conducted.

[0035] Depending upon the size of the sample which is taken from thebody, the collected cells within the specimen could be processed on astandard flow cytometer even prior to initial observation under thefluorescence microscope. In the case of large volumes of fluid containedwithin a cell culture, such as one would obtain on a screen for uterinecervix or colon cancer, a flow cytometer can process large volumes ofcells in a very short period of time. Commercially available flowcytometers may be used. Manufacturers of such devices include Coulter,Becton-Dickenson, and Cytomation. As well understood in the art, flowcytometry involves the suspension of individual cells in a solution,then moving the cells through a tubular system which only allows onecell at a time to flow. The cells pass through a chamber in the systemwhere there is a selection of lasers of selected different frequenciesof light to conduct a number of measurements to include cell counts,cell measurements as to overall size and other parameters. As applied tothe method of this invention, the flow cytometer would have a selectionof lasers which provide light to match the excitation frequency of theparticular compound used to produce fluorescence in the cell sample. Bythis fluorescent tagging, the flow cytometer is able to accurately countthe number of cells which fluoresce. In order to actually separatefluorescing versus non-fluorescing cells, the FACS must be conducted.The FACS involves the inducement of a charge (positive or negative) onthe cell surface of each cell which passes through the flow cytometer.By this induced charge, the fluorescing cells are separated fromnon-fluorescing cells. Accordingly, the fluorescing cells would beplaced into a separate container from the non-fluorescing cells. Asdiscussed above, this concentrated sample of fluorescing cells makesviewing easier under a fluorescence microscope, and for easierphotographing of the fluorescing cells. Because the use of a flowcytometer and the additional step of conducting a FACS involves the useof sophisticated and fairly expensive equipment, in most cases, it isdesirable first to attempt to locate fluorescing cells by simply viewingthem under a fluorescence microscope. However, it shall be understoodthat the method of this invention is not limited to any particularsequence in terms of using a flow cytometer, a fluorescence microscope,or conducting a FACS. Thus, it is conceived within the spirit and scopeof this invention that a FACS could be conducted immediately after thecell sample was centrifuged. However, since the great majority of testsconducted will yield a conclusion of no cancer, it is not advisable toimmediately move to either flow cytometry or conducting a FACS withoutfirst manually observing the cell sample through a fluorescencemicroscope. The capabilities of flow cytometers and fluorescenceactivated cell sorters are well known to those trained in the field.

[0036] As also shown in FIG. 1, introduction of the desired chemicalcompound for purposes of creating fluorescing cells can alternatively beachieved by introducing the compound to the patient prior to cellcollection. This option is illustrated at block 11. As discussed above,the compound 5-ALA may be introduced to the patient orally, topically,or parenterally. The other compounds listed may only be givenparenterally or topically.

[0037] The collected cells may be also placed in culture for observationover a longer period of time. In general, normal non-malignant cellswill survive a much shorter period of time in comparison to malignant orneoplastic cells. This extended observation of the collected cells canbe used as a confirmative test of the initial screening diagnosis.

[0038] Generally speaking, individual physician offices do not havefluorescence microscopes or other equipment which would be used toanalyze the cells in the cell cultures. Accordingly, the cell cultureswould be transported to a regional laboratory for examination by alaboratory technician and a physician. These individuals now performstandard screenings for conventional Pap smears and other cancerscreening procedures.

[0039] As disclosed in Kennedy, et al., U.S. Pat. No. 5,211,938, 5-ALAis a significantly different compound compared to standard porphyrins inthat it, by itself, 5-ALA is not a fluorescent marker, but is aprecursor to a fluorescent marker, namely, protoporphyrin IX. When 5-ALAis administered, it enters a metabolic pathway within the cell and isconverted to PPIX, which is an immediate precursor of heme. 5-ALA may beadministered orally, topically, or by parenteral administration. 5-ALAis taken up by virtually all nucleated cells, and quickly enters intothe “heme” synthesis pathway, eventually resulting in the transformationinto PPIX. In non-malignant cells, the process is blocked by a built-incellular feedback mechanism which effectively stops all PPIX formation.However, the feedback mechanism in malignant cells and those in rapidcell division (characteristic of many pre-malignant cells) is notoperational, and PPIX is produced in significant quantities. 5-ALA isavailable from a manufacturer, Sigma Chemical Co., St. Louis, Mo. At thepresent time, 5-ALA is sold as a reagent worldwide, and not as a USFDA(Food and Drug Administration) approved drug; however, this may changesince applications are in process to use 5-ALA in photo therapy.

[0040] Flow cytometers may also be used not only to count fluorescingcells, but also to measure various dimensions of cells including overallcell diameter, nuclear size, chromatin material in nucleus, cytoplasmicstructures such as mitochondria, and others. Cells may thus be sorted onthe basis of overall cell size. Flow cytometers can also be programmedto count cells of a specific size, and provide overall counts of suchcells in a total specimen. A flow cytometer can also be programmed tosort out and tabulate data on any type of cell from a whole host ofcells in various cell suspensions. It is also understood by thoseskilled in the art that flow cytometers can do other sophisticatedstudies on the cells such as ploidy studies to determine the chromatincontent of the nuclei (diploid cells have the correct number and size ofchromosomes while aneuploid cells are those that have significantalterations in chromosome content). These very exacting measurements canbe done at rates of thousands of cells per second.

[0041] In some circumstances, it may be necessary to provide somepre-separation of the collected cells. For example, based upon thecollection techniques discussed above, some cells will be removed inclumps or sheets of cells. In order to ensure that these clumps orsheets of cells are completely exposed to the compound used as thefluorescent marker, the cells must be separated. Well known laboratoryprocedures exist for separation of these clumps or sheets of cellsthrough use of certain enzymes, chelating agents, and even mechanicalseparation by high speed centrifugation followed by dilution in lowviscosity solutions. These methods of cell “disaggregation” are wellknown in cell sorting laboratories.

[0042] Once the fluorescing cells are identified, the cells can be savedfor further examination by a trained pathologist. Ultimately, apathologist makes a diagnosis as to the presence of a malignancy. If itis determined that the collected cells are malignant, the prior artmethods may then be used to locate and remove the tumor (such asdescribed in Kennedy, et al., U.S. Pat. No. 5,211,938), or the tumor maythen be located and removed by fluorescence guided endoscopic surgery,utilizing endoscopes and surgical devices under guidance offluorescence. If it is determined that open surgery is required toremove a tumor, fluorescence assisted surgery can also be conductedunder these circumstances wherein the surgeons utilize headlightscapable of delivery of tuned frequencies of light, and other lightemitting equipment may be used, such as retractors, probes, anddissectors with built-in illumination.

[0043] The method of this invention primarily utilizes cells whichnormally desquamate from the various surfaces of the body, both internaland external, thus their removal by non-invasive means does not resultin undue patient trauma. These non-invasive cell collection methodsaccelerate the release of such cells from the surfaces of the tumormass, and then, as described above, are collected and analyzed.

[0044]FIGS. 2A and 2B illustrate organizational diagrams of the types ofcells which may be removed according to these non-invasive and minimallyinvasive methods, and also shows the major steps or actions which areused to collect the types of cell. As shown, cell collection 10 may beachieved with cells from the pulmonary system 30, prostate gland 32,cervical/uterine area 34, breast 36, oral areas 38, urinary tract 40,gastrointestinal tract 42, central nervous system 43, and amiscellaneous group 45.

[0045] The specific types of cells which may be exfoliated are asfollows:

[0046] 1. Pulmonary system 30—trachea, bronchi, bronchioli, and alveoli.

[0047] 2. Prostate gland 32—seminal vesicles and ejaculatory ducts.

[0048] 3. Cervical/uterine area 34—uterus, cervix and uterine cavitycells.

[0049] 4. Breast 36—ductal carcinoma cells.

[0050] 5. Oral 38—all cell types exposed in the mouth to include cheeklining, tongue, floor and roof of the mouth, gums and throat.

[0051] 6. Urinary tract 40—kidney, pelvis, calyces, ureters, urinarybladder, and urethra.

[0052] 7. Gastrointestinal tract 42—esophagus, stomach, small intestine,and large intestine (colon).

[0053] 8. Central nervous system 43—ventricles and meninges.

[0054] In addition to the general cell categories discussed above, thereare other cells within the body which may be removed by the non-invasiveor minimally invasive techniques. This miscellaneous group is shown asgroup 45 which includes cells from the peritoneal cavity 64 such asliver, pancreas, ovaries, and other peritoneal cells, and cells from thethoracic cavity 66 such as pleurocentesis cells. Also included from thismiscellaneous group are cells within the sinus and nasal system 68 andauditory canal 70.

[0055] Now, a discussion will follow which more particularly points outthe non-invasive and minimally invasive techniques of cell collectionfor each of the groups of cells.

[0056] Block 44 lists the major actions conducted in collecting cellsfrom the pulmonary system 30. As shown, this non-invasive collectiontechnique includes fist percussion while a patient is placed in apostural drainage position, and then postural drainage allows cell ladenrespiratory fluid to be captured. Sputum, by itself, does not generallycontain pulmonary cells which come from the lining of the air ductswithin the lung (bronchi and alveoli cells). Therefore, the posturaldrainage in conjunction with the fist percussion helps to dislodgepulmonary cells to create a cell laden fluid with bronchi, alveoli, andthe other cells listed above. The patient is placed on the edge of a bedor other horizontal surface with the hips and legs horizontal, and thechest and head hanging down to the floor. This postural drainageposition allows secretions from the alveoli, bronchi, and trachea toflow by gravity through the trachea toward the mouth. Repeated fistpercussion over the entire chest wall helps to continue the dislodgementof cells and to continue the flow through the trachea to the mouth.Coughing at intervals also allows for collection of large numbers ofcells during postural drainage.

[0057] For exfoliation of prostate cells 32, the non-invasive collectionmethod is achieved simply by “milking” the prostate gland. This is shownat block 46. A physician would simply conduct a digital rectalexamination to determine the status of the gland. The gland is “milked”by squeezing the gland to express contained fluids. These fluids arecarried by the ductal system to the urethra via the seminal vesicles andejaculatory ducts.

[0058] For exfoliation of cervical and uterine cells 34, non-invasivecollection can be carried out by use of a cytology brush, shown at step48. One example of a cervical collection brush which can be used isdisclosed in my earlier U.S. Pat. No. 4,762,113. The cytology brushdisclosed in this patent has proven to be superior to standardcytological brushes in its ability to collect the necessary quantity ofcells.

[0059] For exfoliation of cells from the breast 36, the patient isplaced in a face down position with both breasts dropping down throughapertures in an examination table top. This allows both breasts to hang,unsupported, through the table top. The ductal system of the breasts canthen be opened by use of a product such as “Seruminex™.” Gravity allowsfluids retained within the breast ductal system to drain out through theducts in the nipple. To accelerate fluid removal, a commerciallyavailable breast pump could be used, such as that used by nursingmothers. This non-invasive method is shown at block 50.

[0060] In order to obtain cells from the mouth or throat area, acytological brush may be used which brushes the suspicious areas. Thisnon-invasive technique is shown at block 52. The cervical collectionbrush mentioned above is also ideal for use in removal of cells in themouth and throat.

[0061] In order to exfoliate cells from the urinary tract 40, rapid oralfluid intake in conjunction with administration of diuretics such asLasix™ results in cell exfoliation from the transitional cell linings ofthe urinary tract. Fluids are collected from the urethra, and somefluids may be washed out further by the act of urination. This veryrapid flow, occurring at the level of the renal pelvis and continuingthrough the ureters, bladder, and urethra will dislodge cells in largequantities. The cells can then be collected, concentrated bycentrifugation, and preserved in the cell transport media of the cellculture. This method is shown at block 54.

[0062] In order to collect cells from the gastrointestinal tract 42,lavage cytology is utilized by first giving the patient an orallyadministered balanced electrolyte solution. The solution may includedrugs which increase bowel evacuation, such as biscodyl, colyte, andgolytely. This first application of an electrolyte solution induces acleansing wash of the bowel to remove fecal materials. Then, anadditional orally administered electrolyte solution can be given to thepatient to produce a clear anal effluent for cytological evaluation.This clear fluid contains thousands of cells, and if a malignancyexists, cancer cells will be washed out with the large volume of fluid.This technique is shown at block 56.

[0063] Cells can be collected from the central nervous system 43 byconducting a “tap” of the spinal canal with a syringe and needle used instandard spinal tap procedures. Thus, for this type of cell collection,non-invasive means for cell exfoliation is not possible. This techniquefor removal of cells from the central nervous system 43 is shown atblock 72.

[0064] Non-invasive means are also not available for removal of cellsfrom both the chest (thoracic) cavity 66 and the peritoneal cavity 64. Aminimally invasive means of cell collection for these types of cells maybe achieved by utilizing a syringe and needle or using a peritoneallavage catheter. A syringe and needle or a catheter inserted through aneedle is placed in the cavity. Fluids are introduced into the cavity todislodge cells, and then the cell laden fluid is removed from the cavityfor collection. This involves the use of normal saline irrigations viathe catheter (a wash of peritoneal cells). As necessary, the cells arecentrifuged and immediately placed in the cell transport media. Thistechnique is identified as blocks 76 in FIG. 2.

[0065] To induce exfoliation for cells in the auditory canal 70 or thenasal area and sinus passages 68, a cytologic brush may be used. Thebrush is inserted into the ear or nose and placed in contact with thetargeted area. This method is shown at blocks 74 of FIG. 2B. Thecervical cytological brush mentioned above may also be used to collectcells from these areas.

[0066] One very clear example of the advantages of the method of thisinvention over standard cancer screening procedures is with relation toscreening for prostate cancer. The typical procedure for prostate cancerscreening is for the urologist to conduct a digital rectal examinationwhich amounts to a “feel” of the prostate for areas of increasedhardness within the gland. There are very many other reasons for hardlumps in the prostate gland including stones, fibrosis from priorinfection, cysts, and infarction from loss of blood supply to a givensegment of the gland. In addition, the urologist may order a study ofacid phosphatase levels (a substance which may be elevated in thepresence of a prostate cancer), and may order a study of prostatespecific antigen levels (PSA) levels. Pelvis and abdominal x-rays may beused to determine if there are signs of bone metastasis. Ultrasoundstudies may be used to determine if there are any suspicious areas inthe prostate gland. Each of the foregoing studies are presumptive tests,but none are absolutely diagnostic of prostate cancer. Even when thesetests are performed, multiple needle biopsies are done which attempt tofind cancerous areas in the gland. In short, the above-identifiedprocedures are costly, can cause trauma to the patient, and do notnecessarily provide for an early diagnosis. By comparison, the method ofthis invention is a relatively absolute diagnosis of a cancer. Althougha pathologist may still wish to confirm the results of the screeningtest of this invention, the screening test of this invention greatlyeliminates many costly procedures and greatly streamlines early cancerdiagnosis.

[0067] In another aspect of the present invention, in addition to havingthe capability to screen cancer via fluorescence detection, it wouldalso be desirable to simultaneously conduct the cancer screeninginvestigation through another means which tags or identifies potentiallymalignant cells. In accordance therewith, the present invention alsoincludes attaching a radioactive tag to the 5-ALA or other photosensitive compound used, thereby providing medical personnel not onlythe ability to conduct the cancer screening by fluorescence detection,but also through a selected form of tomography.

[0068] The radioactive tag, which may be attached to the photo sensitivecompound, includes such radio isotopes such as ⁶⁴Cu or ⁶⁷Cu. Theseradioisotopes are tightly complexed with most porphyrins. Additionally,certain radioisotopes of zinc are also tightly complexed to porphyrins.

[0069] If a complex such as PPIX-CU is administered to a patient havinga suspected malignancy, the PPIX localizes in malignant cells, carryingthe radioactive tag along. Since the malignant cells preferentiallyaccept the PPIX, there is also the automatic acceptance of theradioactive copper molecule as well. Once the cancer screening has takenplace through the non-invasive or minimally invasive means, the cellswhich do not exit the body due to the exfoliation process are leftbehind and are double tagged with fluorescence as well as radioactivity.

[0070] One reference which discloses a method of using radio taggedporphyrins is U.S. Pat. No. 5,391,547 to Cole et al. This reference ishereby incorporated by reference in its entirety.

[0071] The ⁶⁷Cu provides a source of beta radiation for destruction ofmalignant tissue through radiation therapy, and ⁶⁷Cu also emits gammaradiation which can therefore be used in SPECT. For ⁶⁴Cu, this lighterisotope of copper is a positron emitter, and although it does notprovide the opportunity for radiation treatment, ⁶⁴Cu is a positronemitter which enables screening by PET techniques.

[0072] A PET or SPECT scan, done in addition to analysis of thefluorescening tissue, provides further substantial evidence regarding amalignancy, and the exact location of the major cancer site as well asmetastatic sites. This radioactive tagging of the malignant tissue alsoprovides the ability for a surgeon to better conduct a fluorescence typeendoscopic procedure, because the PET or SPECT scan provides detailedmapping of suspected malignant areas. Thus, in all aspects of conductinginvestigational procedures, as well as treatment, the combination of afluorescent tag and a radioactive tag is advantageous.

[0073] The metaloporphyrin complexes of the present invention can beadministered to a patient alone or in combination with pharmaceuticallyacceptable carriers, the proportion of which is determined by thesolubility and chemical nature of the compound and standardpharmaceutical practice. The physician will determine the ultimatedosage of these agents which will be most suitable for prophylaxis ortreatment and will vary with the form of administration and theparticular compound chosen, and also, with the particular patient undertreatment. The physician will generally aim to initiate treatment withthe lowest dose that will be cytotoxic to the malignant or pre-malignantcells. For the radioactive compounds of the present invention, the doseis measured by the radioactivity emitted by the compound. Thus, theweight of the metaloporphyrin and the coordinated metal is not used indetermining the proper treatment dosage. For most patients, the range ofdosages which effectively kill any malignant or premalignant cells in asite selective manner is between about 5 Gy and 25 Gy of radiation.Typically, the range is between about 9 Gy and about 18 Gy. Preferably,the range is between about 15 Gy and about 20 Gy.

[0074] One example of an investigational procedure which lends itselfwell to the method of this invention would be screening for a suspectedbowel cancer. First, the screening procedure would be conducted byadministering to the patient 5-ALA, and then using a non-invasive methodto check exfoliated cells for fluorescence. If fluorescence is observed,malignant cells left inside the colon are tagged with both thefluorescent marker as well as the radioactive tag. PET scanning or SPECTscanning could then be conducted to determine the exact location of anysites of cancer located not only the bowel, but throughout the entirebody. These locations would include the basic cancer site in the bowel,as well as additional sites of extension of the tumor, for example,through the wall of the bowel, and any other metastatic sites such as inthe liver, lymph nodes, and lung. Once the scan has been reviewed, it isthen possible to pass a fluorescence endoscope into the bowel to findthe specific locations shown on the scan.

[0075] Another example of a type of cancer which is well suited foridentification and treatment by the method herein is breast cancer. Theintroduction of 5-ALA to the patient tagged with a radioisotope can beused to not only examine cells from the breast removed by exfoliation,but can also provide a scan of both breasts outlining any sites ofprimary cancer. A surgeon looking for the tumor or tumors has theadditional advantage of having fluorescence of the lesions which wouldaid the surgeon in locating and resecting all tumor sites.

[0076] Use of a dual tagging mechanism has a number of options in termsof not only identification of malignancies, but also treatment options.Referring now to FIG. 3, some of these investigational and treatmentoptions are illustrated. Beginning first with block 100, the patient isprovided the radio tagged compound. The specified period of time is thengiven to allow cell uptake of the compound, shown at block 102. In block104, the cell collection is undertaken utilizing either the non orminimally invasive methods. The exfoliated cells are then examined by afluorescence microscope shown at block 106. At block 108, iffluorescence is observed, then tomographic scanning can be conducted,shown at block 110. If there is no fluorescence observed, then flowcytometry can be conducted, shown at block 112. If at decision block114, fluorescence is observed after conducting the flow cytometry, thenthe tomographic scanning can be conducted, as shown at block 110. If nofluorescence is observed, then there is presumptive conclusion of nomalignancy, shown at block 116.

[0077] If the scanning has taken place, the treating physician then hasa number of options. One option would be to conduct fluorescence guidedendoscopy in order to further pinpoint the location of the malignancies,as shown at block 118. During the same procedure, the doctor would alsohave the option of conducting radiation therapy, shown at block 120, andof course, the doctor could also conduct the radiation therapy withoutfirst conducting the fluorescence guided endoscopy. The treatingphysician would also then have the option of conducting the necessarysurgical procedure, shown at block 122, and/or the physician couldsimply conduct further observation, shown at block 124.

[0078] A number of variations to the methodology shown in FIG. 3 can beused to achieve useful results in combining a fluorescence tracer andradio tracer. For example, tomographic scanning could be conducted priorto fluorescence observation. Thus, the scanning would be considered thefirst phase in the investigation and the fluorescence observation wouldbe considered the second or confirmatory phase in the investigation.

[0079] Utilizing two separate means for identification and treatment ofa cancer has the many advantages as discussed above. The use of acompound which results in fluorescing cancerous tissue allows medicalpersonnel to pinpoint cancerous areas, and provides many options intreating the cancer, which would include not only photo-dynamic therapy,but other traditional modes of treatment. Attaching a radioactive tag tothe compound provides yet another dimension for identification andtreatment of the cancer. The radioactive tag further allows medicalpersonnel to accurately locate, and then provide additional treatment.The advances in SPECT and PET scanning enables a treating physician tomap and record the coordinates of cancerous tissue for subsequenttreatment.

[0080] This invention has been described in detail with reference toparticular embodiments thereof, but it will be understood that variousother modifications can be incorporated within the spirit and scope ofthe invention.

What is claimed is:
 1. A method of cancer screening comprising the stepsof: administering a compound to a patient for uptake into target cells,the compound having a fluorescent marker portion, and a radioactiveportion in target cells, wherein said fluorescent marker portion isselected from the group consisting of 5-ALA, protoporphyrin IX, TCPP,hematoporphyrin derivative, photofrin, uroporphyrin, coproporphyrin,TPPS, and TMPP, and said radioactive portion is a radioisotope ofcopper; collecting the targeted cells from the patient; stimulating thecollected targeted cells with a frequency of light that will causepre-malignant and malignant cells to fluoresce; conducting tomographicscanning of the patient after the stimulating step if fluorescenceoccurred; and conducting radiation therapy by site selective ionizingradiation treatment from said radioactive portion of said compoundwherein the compound is administered in a dose emitting between about 5Gy and 25 Gy radiation.
 2. A method, as claimed in claim 1, furtherincluding the step of: conducting fluorescence guided endoscopy forfurther observation or treatment of areas found during the firstconducting step.
 3. The method of claim 1, wherein the compound isadministered in a dose emitting between about 9 Gy and about 18 Gyradiation.